In recent years, the incidence of life threatening mycoses and the range of fungal species causing these infection have been increasing globally. Although AIDS accounts for much of this increase, other contributing factors include the expanded use of immunosuppressive agents to treat patients with transplants or cancer. The most prevalent opportunistic mycoses are caused by species of Candida, Cryptococcus neoformans, and a variety of hyphomycetes. As a result of this mycological crisis, there is a pressing need to improve the accuracy and speed of the diagnosis, to identify the sources of individual cases and outbreaks, and to understand the genetics and distribution of populations of pathogenic fungi. This proposal will further develop DNA-based approaches to expedite the diagnosis of fungal infections, to investigate genetic variation within populations of Candida albicans, and to identify individual strains, all of which will facilitate efforts to study the epidemiology of outbreaks, the sources of pathogens, and the recognition of strains with pathobiological features, such as drug resistance, tissue tropism or enhanced virulence. During the previous three year period of support, significant progress was achieved. The method of PCR fingerprinting was developed to identify individual strains of C. albicans, Candida krusei, and C. neoformans. PCR fingerprinting should be applicable to a wide variety of fungal pathogens and other eukaryotes. Four inter-related specific aims are proposed: 1) To evaluate the stability and utility of PCR fingerprinting of medical fungi. 2) To develop DNA based resources and methods for the clinical mycology laboratory. 3) To investigate the genetics of populations of C. albicans to understand the exchange of genetic information in this asexual diploid species. 4) To compare relevant sequences of the ribosomal DNA and other genes to clarify the phylogeny of the genus Candida. The results will also benefit basic mycological research by providing the tools to genotype isolates under investigation.